ABSTRACT The provision of antiretroviral therapy (ART) for over 20 million HIV-infected people has been a global success, but the persistence of HIV-associated tuberculosis (TB) and emergence of drug-resistant HIV have presented major barriers to reducing mortality. First, TB is the leading cause of mortality among people living with HIV worldwide, due in part to inadequate diagnostic and prognostic tests. In South Africa, we have conducted three clinical studies to demonstrate that a TB-specific glycolipid, lipoarabinomannan (LAM), can identify some HIV- infected adults with active and subclinical (asymptomatic, culture-confirmed) TB disease, and predict mortality. However, our clinic-based studies found the first point-of-care (POC) urinary LAM assay to have limited diagnostic sensitivity. This prompted us to develop a more sensitive, second-generation urinary LAM assay, which is now ready for clinical validation. In addition, HIV drug resistance mutations in people starting or restarting antiretroviral therapy (pre-treatment drug resistance, PDR) is associated with poorer virological outcomes on first-line non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimens. Our collaborative group has recently documented a sharp rise in PDR in two hyper-endemic rural districts of KwaZulu-Natal, South Africa. We have also developed a simple, inexpensive clinic-based assay to identify common genotypic drug resistance mutations, which is also ready for a clinical validation study. Our objective for this proposal is to validate a second-generation urinary LAM assay for HIV-associated TB and a clinic-based assay for HIV PDR within our biorepository of well-characterized specimens from HIV-infected adults in South Africa. Our central hypotheses, based on preliminary data, are that both a second-generation urinary LAM assay and a novel HIV drug resistance assay will be valid in a South African cohort of HIV-infected adults. We will test our central hypotheses via two specific aims: (1) to validate a second-generation urine LAM assay in adults with HIV-associated active and subclinical TB disease in KwaZulu-Natal, South Africa; (2) to validate a clinic-based assay for the detection of key HIV drug resistance mutations, including minority variants associated with virological outcomes, in the same cohort of HIV-infected adults. To accomplish these aims, we will leverage our existing biorepository of well- characterized clinical specimens to evaluate a second-generation urine LAM assay and HIV genotypic resistance testing using a rapid PDR assay. Our study will be novel for validating a rapid, second-generation POC urinary LAM assay for HIV-associated TB and a clinic-based assay for key HIV drug resistance mutations, including minority variants, in a cohort of HIV-infected adults in South Africa.